Spring 2021

Phylogenetic tree

Three life domains: bacteria, archaea, and eukaryota

  • Bacteria are a type of biological cell. They constitute a large domain of prokaryotic microorganisms
  • Archaea constitute a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes
  • Eukaryotes are organisms whose cells have a nucleus enclosed within a nuclear envelope

Overview

  • Cells are the fundamental units of all living organisms
  • Each cell is a complex system consisting of many substructures
    • Viruses are simplest organisms (∼ 10,000 bp. long genomes), which require a living host
    • Prokaryotes are simplest free-living organisms, e.g. bacteria (∼ 1,000,000 bp. long genomes)
    • Eukaryotes have cells which contain internal structures such as a nucleus, e.g. yeast
    • Multi-celled organisms involve cell specialization, requiring differential gene expression and inter-cellular signaling

Comparing the Sizes of Microorganisms, Cell Size and Scale

Procaryotic cell

Eukaryotic cell

Differences between Prokaryotic and Eukaryotic Cells

The genome: the secret of life

Your genome, along with your environment and experiences, shapes who you are

  • Height
  • Hair, eye, skin color
  • Broad/narrow, small/large features
  • Susceptibility to disease
  • Response to drug treatments
  • Longevity and cognition

Physical traits tend to be strongly genetic

Eukaryotic cells and the genome

  • Each cell contains a complete copy of an organism’s genome, or blueprint for all cellular structures and activities

  • The genome is distributed along chromosomes, which are made of compressed and entwined DNA

  • Cells are of many different types (e.g. blood, skin, nerve cells), but all can be traced back to a single cell, the fertilized egg

The nucleus

  • The nucleus is a sub-compartment found only in eukaryotic cells, in which the organism’s DNA resides

  • The nuclear membrane separates the nucleus from the rest of the cell, which is called the cytoplasm

  • The entire cell is enclosed by the plasma membrane

  • Embedded within this membrane is a variety of protein structures that act as channels and pumps to control movement into and out of the cell

Discovery of chromosomes

  • By the mid-1800s, microscopes were powerful enough to observe the presence of unusual structures called “chromosomes” that seemed to play an important role during cell division.

  • It was only possible to see the chromosomes unless appropriate stains were used

  • “Chromosome” comes from the Greek words meaning “color body”

Drawing of mitosis by Walther Flemming. Flemming, W. Zellsubstanz, Kern und Zelltheilung (F. C. W. Vogel, Leipzig, 1882).

Chromosomes

  • Chromosomes are packets of compressed and entwined DNA and are located in the nucleus
  • Each chromosome carries its own unique set of genes. The specific site along a specific chromosome that a gene is located is called its genetic locus
  • Haploid (one copy) human genome has 23 chromosomes, autosomes (chromosome 1-22) and one sex chromosome (X, Y)
  • Human genome is diploid - comprised of a paternal and a maternal “haplotype”. Together, they form our “genotype” of 46 chromosomes
  • Germ cells, sperm and egg, carry only a single copy of each chromosome and gene and are called haploid

The basic structure of a chromosome

  • Size. This is the easiest way to tell chromosomes apart

  • Banding pattern. The size and location of Giemsa bands make each chromosome unique

  • Centromere position. Centromeres appear as a constriction. They have a role in the separation of chromosomes into daughter cells during cell division (mitosis and meiosis)

http://learn.genetics.utah.edu/content/basics/readchromosomes/

Chromosome Giemsa banding (G-banding)

  • Heterochromatic regions, which tend to be rich with adenine and thymine (AT-rich) DNA and relatively gene-poor, stain more darkly with Giemsa and result in G-banding

  • Less condensed (“open”) chromatin, which tends to be (GC-rich) and more transcriptionally active, incorporates less Giemsa stain, resulting in light bands in G-banding

https://ghr.nlm.nih.gov/chromosome/1#ideogram

Chromosome Giemsa banding (G-banding)

  • Cytogenetic bands are labeled p1, p2, p3, q1, q2, q3, etc., counting from the centromere out toward the telomeres. At higher resolutions, sub-bands can be seen within the bands

  • For example, the locus for the CFTR (cystic fibrosis) gene is 7q31.2, which indicates it is on chromosome 7, q arm, band 3, sub-band 1, and sub-sub-band 2. (Say 7,q,3,1 dot 2)

https://ghr.nlm.nih.gov/chromosome/1#ideogram

The role of the centromere

  • Centromeres are required for chromosome separation during cell division
  • The centromeres are attachment points for microtubules, which are protein fibers that pull duplicate chromosomes toward opposite ends of the cell before it divides
  • This ensures that each daughter cell have a full set of chromosomes
  • Each chromosome has only one centromere

http://learn.genetics.utah.edu/content/basics/readchromosomes/

Centromere positions

The position of the centromere relative to the ends helps to tell chromosomes apart

  • Metacentric - the centromere lies near the center of the chromosome
  • Submetacentric - the centromere that is off-center, so that one chromosome arm is longer than the other. The short arm is designated “p” (for petite), and the long arm is designated “q” (because it follows the letter “p”)
  • Acrocentric - the centromere is very near one end

http://learn.genetics.utah.edu/content/basics/readchromosomes/

Building blocks of DNA

  • The basic unit (nucleotide) is composed of an organic base attached to a deoxyribose sugar

  • The phosphate group also attached to the sugar

  • The base is one of cytosine (C), thymine (T), adenine (A), and guanine (G)

Bases

The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T)

Discovery of double helix, 1953

  • James Watson and Francis Crick

Genomic DNA

Before the discovery

Genomic DNA

  • DNA is a double helix, with bases to the center (like rungs on a ladder) and sugar-phosphate units along the sides of the helix (like the sides of a twisted ladder)
  • The strands are complementary (Watson-Crick base pairing rules)
  • A (purine) pairs with T (pyrimidines) C (pyrimidines) pairs with G (purine)
  • The pairs held together by hydrogen bonds. The helix is caused by the use of the hydrogen bonds between the single-strands

Base pairing

  • The force that holds a base pair together is a weak hydrogen bond

  • Although each individual bond is weak, their cumulative effect along the strands is strong enough to bind the two strands tightly together

  • As a result, DNA is chemically inert and is a stable carrier of genetic information

  • Rules of base pairing: A-T(U), C-G

Eukaryotic DNA packaging

Genes

  • Genes are discrete hereditary units located on the chromosomes

  • Genes are segments of deoxyribonucleic acid (DNA) that contain the code for a specific protein that functions in one or more types of cells in the body

  • Genes contain information used to synthesize proteins, the building blocks of cells

The central dogma of molecular biology

  • Formulated by Francis Crick in 1956

  • DNA makes RNA and RNA makes protein

  • Transcription is the making of an RNA molecule off a DNA template

  • Translation is the making of a protein off an RNA template

RNA vs. DNA: Single vs. double strands

Nucleic acids - RNA

  • Messenger RNA (mRNA) - carrier of genetic information

  • Transfer RNA (tRNA) - deliver amino acids for protein synthesis

  • Ribosomal RNA (rRNA) - central component of ribosome, protein manufacturing machinery

  • Small RNA (siRNA, miRNA, snRNA, piwiRNA) - regulation of transcription/translation

Transcription

  • In transcription, the DNA double helix opens along its length
  • One strand of the open helix remains inactive, while the other strand acts as a template against which a complementary strand of mRNA forms
  • The sequence of bases along the mRNA strand is identical to the sequence of bases along the inactive DNA strand, except uracil (U) replaces T. Also RNA has ribose sugar instead of deoxyribose sugar.
  • RNA transcribed from 5’ to 3’ end

Gene structure in prokaryotes

Genes of related function are often organized and transcribed together under the control of a single promoter

Gene structure in eukaryotes

  • Non-coding interruptions are known as intervening sequences or introns
  • Coding sequences that are expressed are exons
  • Most, but not all eukaryote genes contain introns. Although transcribed, these introns are excised (cut out) before translation

Intron boundaries

  • Introns always have two distinct nucleotides at either end.
  • At the 5’ end the DNA nucleotides are GT [GU in the premessenger RNA (pre-mRNA)]; at the 3’ end they are AG.
  • These nucleotides are part of the splicing sites.

The GT/AG mRNA processing rule is applicable for almost all eukaryotic genes

http://www.imgt.org/IMGTeducation/Aide-memoire/_UK/splicing/

Alternative splicing

mRNA splicing - variants of mRNA assembly

  • Tissue specific alternative splicing patterns of the \(\alpha\)-tropomyosin pre-mRNA molecule
  • Exons are blue rectangles
  • Introns are black carets

Untranslated regions (UTRs)

  • The 5’ UTR is upstream from the coding sequence
    • Within the 5’ UTR is a sequence that is recognized by the ribosome which allows the ribosome to bind and initiate translation
    • The 5’ UTR of Eukaryotes contains a Kozak sequence (ACCAUGG)
  • The 3’ UTR is found immediately following the translation stop codon
    • The 3’ UTR plays a critical role in translation termination as well as post-transcriptional modification
    • Contains regulatory regions, AU-rich elements
    • Size ranges from 60 to 4,000 bases (average 800)

RNA processing

  • The RNA transcript is extensively modified before export to the cytoplasm
  • A cap of 7-methylguanine (m7G, a series of an unusual base) is added to the 5’ end of the mRNA. This cap is essential for binding the mRNA to the ribosome
  • A string of adenines (as many as 200 nucleotides known as poly-A tail) is added to the 3’ end of the mRNA after transcription. The function of a poly-A tail is not known, but it can be used to capture mRNAs
  • Introns are cut out of the message and the exons are spliced together before the mRNA leaves the nucleus

http://vcell.ndsu.nodak.edu/animations/mrnaprocessing/movie-flash.htm

Translation

Gene expression

  • Each cell contains a complete copy of the organism’s genome. A gene that is transcribed is said to be expressed

  • Not all cells express the same genes which is why different cells perform different functions

  • Even within the same cell different genes will be expressed at different times and perhaps at different levels

Housekeeping genes

  • Housekeeping genes are genes that are required for the maintenance of basal cellular functions that are essential for the existence of a cell, regardless of its specific role in the tissue or organism

  • They are expected to be expressed in all cells of an organism under normal conditions, irrespective of tissue type, developmental stage, cell cycle state, or external signal

  • Can be used as internal controls in gene expression studies

Housekeeping genes

  • Typical examples:
    • glyceraldehyde- 3-phosphate dehydrogenase (GAPDH)
    • tubulins (beta-tubulin TUBB)
    • cyclophilin (cyclophilin A CYPA)
    • albumin (ALB)
    • actins (beta-actin ACTB)
    • 18S rRNA or 28S rRNA

3,804 housekeeping genes (and exons) defined from Human BodyMap project gene expression data http://www.tau.ac.il/~elieis/HKG/

Eisenberg, Eli, and Erez Y. Levanon. “Human Housekeeping Genes, Revisited.” Trends in Genetics: TIG 29, no. 10 (October 2013): 569–74. doi:10.1016/j.tig.2013.05.010.

Transcription factors

  • Transcription factors (TFs) are proteins that bind to specific DNA sequences in the control region of each gene and determine whether or not the gene will be transcribed

  • The specific stretch of nucleotide sequence to which the TFs bind, often called a sequence motif, is usually quite short, typically 5-10 nucleotides long

  • Some TFs provide the RNA polymerase enzyme with access to the gene while other TFs block such access to ensure the gene is transcriptionally repressed

Human Genome Project

  • Computational biology attempts to use genome sequence to ascertain function of genes

  • Although genomes vary slightly from person to person, it seemed reasonable to try to establish a consensus human genome sequence

  • Robert Sinsheimer, chancellor of UC Santa Cruz, proposed to sequence the human genome in 1984

  • After much debate, the human genome project started in October 1990

Genome sizes compared

DNA alterations

Changes in DNA sequence/amount alter gene and/or protein product

  • One base being replaced by another (substitution)
  • A base being excised (deletion)
  • A base being added (insertion)
  • A small subsequence of bases being removed and reinserted in the opposite direction (inversion)
  • A small subsequence of bases being removed and reinserted in a different place (translocation)

Hallmarks of cancer

Biotechnologies

  • The Polymerase chain reaction (PCR) enables one to make many copies of a particular DNA sequence anywhere in solution given only the starting/ending sequences (primers)

  • Developed in 1983 by Kary Mullis, PCR allows for the amplification of DNA fragments that are flanked by known “primer” sequences

  • Exquisitely sensitive and specific, it can amplify a single molecule in a sample into billions of copies with nearly perfect fidelity

  • Uses naturally occurring polymerase enzymes that copy DNA by adding free nucleotides to a single-stranded template

Polymerase Chain Reaction (PCR)

Extra

Karyotype

  • Cytogenetics is the study of normal and abnormal chromosomes

  • The normal configuration of chromosomes is often termed the euploid karyotypic state

  • Euploidy implies that each of the autosomes is present in normally structured pairs and that the X and Y chromosome are present in normally structured pairs for the sex of the individual

  • Deviation from the euploid karyotype - the state termed aneuploidy - is some alteration in the overall chromosome structure, such as loss of entire chromosomes, the presence of extra copies of chromosomes, etc.

Karyotype

Cell cycle

Three RNA polymerases

  • RNA polymerase is an enzyme that produces RNA
  1. RNA Pol I - transcription of ribosomal RNA (not the 5S subunit)
  2. RNA Pol II - mRNA, snRNA, microRNA
  3. RNA Pol III - tRNA, 5S rRNA, small RNA

Three stages of transcription

DNA replication

  • In DNA replication, DNA polymerases unwind the DNA molecule by breaking the H-bonds between bases - strands separate

  • Once the polymerases have opened the molecule, an area known as the replication fork forms (always initiated at a certain set of nucleotides, the origin of replication)

  • New nucleotides are placed in the fork and link to the corresponding parental nucleotide already there (A with T, C with G)

DNA replication